Protein synthesis inhibitionassay - (Oct/19/2001 )
I am searching for a protocol for assaying the protein synthesis inhibition in cultured cells using radioactive (3H -Leucine)/non-radioactive method. If anyone has ever done such an assay, kindly send the protocol to my email address.
Incubate cells with 0.5microcuries/ ml of tritiated leucine for 1 hour in leucine free media like RPMI-1640.Then fix the cells in 3%w/v perchloric acid and 0.5%w/vphosphotungastic acid .wash the cells with PBS twice.Transfer the cells to to filter paper using cell harvester and then count.If cell harvester is not available dissolve the cells in 0.5N NaOH Take an aliqout tranfer to scintillation cocktail.Neutralize with HCl.if you don't neutralize the solution is turbid.Take the count
If you are using a cell harvester don't fix the cells.After washing the cells use the cell harvester to transfer the cells.